Bacteria and fungi have a variety of commercial applications and are used in the production of alcoholic beverages, food products, pesticides, food additives, biofuels, probiotics, and pharmaceuticals. Microbes with a combination of phenotypic traits that are not commonly encountered in nature are often required for some applications, such as biotherapeutics. Wild-type microbial strains show evolutionary development for survival and reproduction. However, commercial applications often require strains with a mixture of traits that both optimize the industrial process and produce products in amounts that are often not advantageous within the microbe's natural environment. Strain alteration and improvement methods were developed to create new commodities and/or increase profitability. Traditional strain alteration is dominated by three different approaches, each with its own limitations, as described below.
The first, classical mutagenesis, relies on chemically induced point and shift mutations of existing genes. Most of these mutations are neutral or even deleterious (Eyre-Walker, et al. (2007) Nature Reviews Genetics 8:610-618). The resulting mutations are completely random, and desired strains are found by using selective medium after a mutagen is applied. One limitation is that mutagenesis cannot be used to add exogenous genes from other strains or species. In addition, repeated rounds of mutagenesis can be subject to “Muller's Ratchet,” which is the process by which the genomes of an asexual population accumulate deleterious mutations in an irreversible manner. Each new round of mutagenesis makes finding a viable, improved mutant more difficult due to the accumulation of deleterious mutations. Other strain improvement methods include recombination, which helps to avoid Muller's Ratchet.
The second method of strain alteration is recombination, in which hybrids are formed through techniques such as protoplast fusion. This technique fuses two different strains together by first removing the parents' cell walls and then fusing the resulting protoplasts together through spontaneous or induced fusion. This can create microbes and plants with polyploidy that sometimes have greater productivity than the individual parent strains. This method allows the recombination of strains that may not normally conjugate, and is useful when target genes are unknown or a polygenic trait needs to be transferred. The fusion results in the combination of both parents' entire genomes. It is a method that has been most successfully used for the alteration of crop plants and molds that function well with polyploidy. In some yeast, karyogamy usually occurs a few generations later, whereupon one of the transferred nuclei can be lost before recombination has occurred. Protoplast fusion has been unsuccessful for gram negative bacteria and although there has been some success with protoplast fusion in gram positive bacteria, the success rate is extremely low so it is not often used. There is a low success rate of hybrid formation between strains of the same species, such as Bacillus subtilis (Schaeffer, P. et al (1976) Proc Natl Acad Sci 73(6):2151-2155). Protoplasts were first formed at the rate of 2.5×10−8 from initial wild-type cell cultures. Then, protoplasts were fused to create hybrids at a maximum rate of 4×10−3 per pairing of parent protoplasts. This means the best final frequency of hybrids per one original parent cell is only 6.4×10−12. Combining protoplasts from different species has an even lower success rate and in most cases is not a reliable or economically viable option for horizontal gene transfer in bacteria.
The third method to alter strains is genetic engineering, which has been demonstrated in many successful examples, and which is horizontal gene transfer via a vector. Researchers must know which genes need to be transferred in order to express the desired phenotype. Genetic engineering requires extensive metabolic knowledge, which is often limited by invalid assumptions. Most methodologies require a constructed plasmid containing the excised exogenous gene of interest along with an origin of replication and an appropriate promoter. This plasmid is then cloned and must be inserted into the host species via transformation, transduction, or conjugation, or friction via the Yoshida Effect. Transposons have also been transferred from one species to another in vitro and may be spontaneously incorporated into the chromosomal DNA, but this technique also initially requires an artificial vector to initiate the transfer. Extensive, robust techniques are published for E. coli that result in negligible changes in the genetically modified organism (GMO) growth rate or metabolic function. However, other species are not so well understood and may display faulty gene expression of the inserted plasmid, resistance to the initial transformation, or lose the exogenous plasmid easily. Another major disadvantage is the classification of resulting strains as GMOs, which can limit or exclude some industrial applications.
There is a need for an alternative, non-GMO, economically viable method to perform horizontal gene transfer for the purpose of modifying industrial microbial strains.